Shane Fischbach

M2 macrophages promote beta-cell proliferation by up-regulation of SMAD7

Significance Here, we show how, mechanistically, inflammation-recruited macrophages may stimulate beta-cell proliferation in the pancreas, and specifically identify that TGFβ1 and EGF, which are secreted by M2 macrophages, induce SMAD7 expression in beta cells. SMAD7 not only activates cell cycle activators but also induces the nuclear exclusion of cell cycle inhibitors to promote beta-cell replication. Our study thus reveals a molecular pathway to induce beta-cell proliferation through enhanced SMAD7 activity specifically in beta cells. Determination of signaling pathways that regulate beta-cell replication is critical for beta-cell therapy. Here, we show that blocking pancreatic macrophage infiltration after pancreatic duct ligation (PDL) completely inhibits beta-cell proliferation. The TGFβ superfamily signaling inhibitor SMAD7 was significantly up-regulated in beta cells after PDL. Beta cells failed to proliferate in response to PDL in beta-cell–specific SMAD7 mutant mice. Forced expression of SMAD7 in beta cells by itself was sufficient to promote beta-cell proliferation in vivo. M2, rather than M1 macrophages, seem to be the inducers of SMAD7-mediated beta-cell proliferation. M2 macrophages not only release TGFβ1 to directly induce up-regulation of SMAD7 in beta cells but also release EGF to activate EGF receptor signaling that inhibits TGFβ1-activated SMAD2 nuclear translocation, resulting in TGFβ signaling inhibition. SMAD7 promotes beta-cell proliferation by increasing CyclinD1 and CyclinD2, and by inducing nuclear exclusion of p27. Our study thus reveals a molecular pathway to potentially increase beta-cell mass through enhanced SMAD7 activity induced by extracellular stimuli.

Pancreatic duct cells as a source of VEGF in mice

Aims/hypothesisVascular endothelial growth factor (VEGF) is essential for proper pancreatic development, islet vascularisation and insulin secretion. In the adult pancreas, VEGF is thought to be predominantly secreted by beta cells. Although human duct cells have previously been shown to secrete VEGF at angiogenic levels in culture, an analysis of the kinetics of VEGF synthesis and secretion, as well as elucidation of an in vivo role for this ductal VEGF in affecting islet function and physiology, has been lacking.MethodsWe analysed purified duct cells independently prepared by flow cytometry, surgical isolation or laser-capture microdissection. We infected duct cells in vivo with Vegf (also known as Vegfa) short hairpin RNA (shRNA) in an intrapancreatic ductal infusion system and examined the effect of VEGF knockdown in duct cells in vitro and in vivo.ResultsPancreatic duct cells express high levels of Vegf mRNA. Compared with beta cells, duct cells had a much higher ratio of secreted to intracellular VEGF. As a bioassay, formation of tubular structures by human umbilical vein endothelial cells was essentially undetectable when cultured alone and was substantially increased when co-cultured with pancreatic duct cells but significantly reduced when co-cultured with duct cells pretreated with Vegf shRNA. Compared with islets transplanted alone, improved vascularisation and function was detected in the islets co-transplanted with duct cells but not in islets co-transplanted with duct cells pretreated with Vegf shRNA.Conclusions/interpretationHuman islet preparations for transplantation typically contain some contaminating duct cells and our findings suggest that the presence of duct cells in the islet preparation may improve transplantation outcomes.

Pancreatic cell tracing, lineage tagging and targeted genetic manipulations in multiple cell types using pancreatic ductal infusion of adeno-associated viral vectors and/or cell-tagging dyes

Genetic manipulations, with or without lineage tracing for specific pancreatic cell types, are very powerful tools for studying diabetes, pancreatitis and pancreatic cancer. Nevertheless, the use of Cre/loxP systems to conditionally activate or inactivate the expression of genes in a cell type– and/or temporal-specific manner is not applicable to cell tracing and/or gene manipulations in more than one lineage at a time. Here we report a technique that allows efficient delivery of dyes for cell tagging into the mouse pancreas through the duct system, and that also delivers viruses carrying transgenes or siRNA under a specific promoter. When this technique is applied in genetically modified mice, it enables the investigator to perform either double lineage tracing or cell lineage tracing combined with gene manipulation in a second lineage. The technique requires <40 min.

Transient Suppression of TGFβ Receptor Signaling Facilitates Human Islet Transplantation

Although islet transplantation is an effective treatment for severe diabetes, its broad application is greatly limited due to a shortage of donor islets. Suppression of TGFβ receptor signaling in β-cells has been shown to increase β-cell proliferation in mice, but has not been rigorously examined in humans. Here, treatment of human islets with a TGFβ receptor I inhibitor, SB-431542 (SB), significantly improved C-peptide secretion by β-cells, and significantly increased β-cell number by increasing β-cell proliferation. In addition, SB increased cell-cycle activators and decreased cell-cycle suppressors in human β-cells. Transplantation of SB-treated human islets into diabetic immune-deficient mice resulted in significant improvement in blood glucose control, significantly higher serum and graft insulin content, and significantly greater increases in β-cell proliferation in the graft, compared with controls. Thus, our data suggest that transient suppression of TGFβ receptor signaling may improve the outcome of human islet transplantation, seemingly through increasing β-cell number and function.

Epidermal Growth Factor Receptor Signaling Regulates β Cell Proliferation in Adult Mice

A thorough understanding of the signaling pathways involved in the regulation of β cell proliferation is an important initial step in restoring β cell mass in the diabetic patient. Here, we show that epidermal growth factor receptor 1 (EGFR) was significantly up-regulated in the islets of C57BL/6 mice after 50% partial pancreatectomy (PPx), a model for workload-induced β cell proliferation. Specific deletion of EGFR in the β cells of adult mice impaired β cell proliferation at baseline and after 50% PPx, suggesting that the EGFR signaling pathway plays an essential role in adult β cell proliferation. Further analyses showed that β cell-specific depletion of EGFR resulted in impaired expression of cyclin D1 and impaired suppression of p27 after PPx, both of which enhance β cell proliferation. These data highlight the importance of EGFR signaling and its downstream signaling cascade in postnatal β cell growth.

Suppression of microRNA-205-5p in human mesenchymal stem cells improves their therapeutic potential in treating diabetic foot disease

Diabetes is a prevalent disease endangering human health, while diabetic foot disease (DF) is one of the most severe complications of diabetes. Mesenchymal stem cells (MSCs) have been used in DF treatment, taking advantage of the differentiation potential of MSCs into endothelial cells and their production and secretion of trophic factors like vascular endothelial growth factor (VEGF). Molecular modification of MSCs to improve their therapeutic effects has been recently applied in treating other diseases, but not yet in DF. Here, we found that micoRNA-205-5p (miR-205-5p) is expressed in human MSCs, and miR-205-5p inhibits protein translation of VEGF through its interaction with 3′-UTR of the VEGF mRNA. Expression of antisense of miR-205-5p (as-miR-205-5p) significantly increased both cellular and secreted VEGF by MSCs, which significantly improved the therapeutic effects of MSCs on DF-associated wound healing in diabetic NOD/SCID mice. Together, our data suggest that miR-205-5p suppression in MSCs may improve their therapeutic effects on DF, seemingly through augmentation of VEGF-mediated vascularization.

Forkhead Box Protein 1 (FoxO1) Inhibits Accelerated β Cell Aging in Pancreas-specific SMAD7 Mutant Mice

The mechanisms underlying the effects of exocrine dysfunction on the development of diabetes remain largely unknown. Here we show that pancreatic depletion of SMAD7 resulted in age-dependent increases in β cell dysfunction with accelerated glucose intolerance, followed by overt diabetes. The accelerated β cell dysfunction and loss of proliferation capacity, two features of β cell aging, appeared to be non-cell-autonomous, secondary to the adjacent exocrine failure as a “bystander effect.” Increased Forkhead box protein 1 (FoxO1) acetylation and nuclear retention was followed by progressive FoxO1 loss in β cells that marked the onset of diabetes. Moreover, forced FoxO1 expression in β cells prevented β cell dysfunction and loss in this model. Thus, we present a model of accelerated β cell aging that may be useful for studying the mechanisms underlying β cell failure in diabetes. Moreover, we provide evidence highlighting a critical role of FoxO1 in maintaining β cell identity in the context of SMAD7 failure.

PNA lectin for purifying mouse acinar cells from the inflamed pancreas

Better methods for purifying human or mouse acinar cells without the need for genetic modification are needed. Such techniques would be advantageous for the specific study of certain mechanisms, such as acinar-to-beta-cell reprogramming and pancreatitis. Ulex Europaeus Agglutinin I (UEA-I) lectin has been used to label and isolate acinar cells from the pancreas. However, the purity of the UEA-I-positive cell fraction has not been fully evaluated. Here, we screened 20 widely used lectins for their binding specificity for major pancreatic cell types, and found that UEA-I and Peanut agglutinin (PNA) have a specific affinity for acinar cells in the mouse pancreas, with minimal affinity for other major pancreatic cell types including endocrine cells, duct cells and endothelial cells. Moreover, PNA-purified acinar cells were less contaminated with mesenchymal and inflammatory cells, compared to UEA-I purified acinar cells. Thus, UEA-I and PNA appear to be excellent lectins for pancreatic acinar cell purification. PNA may be a better choice in situations where mesenchymal cells or inflammatory cells are significantly increased in the pancreas, such as type 1 diabetes, pancreatitis and pancreatic cancer.

SMAD3/Stat3 Signaling Mediates β-Cell Epithelial-Mesenchymal Transition in Chronic Pancreatitis–Related Diabetes

Many patients with chronic pancreatitis develop diabetes (chronic pancreatitis–related diabetes [CPRD]) through an undetermined mechanism. Here we used long-term partial pancreatic duct ligation (PDL) as a model to study CPRD. We found that long-term PDL induced significant β-cell dedifferentiation, followed by a time-dependent decrease in functional β-cell mass—all specifically in the ligated tail portion of the pancreas (PDL-tail). High levels of transforming growth factor β1 (TGFβ1) were detected in the PDL-tail and were mainly produced by M2 macrophages at the early stage and by activated myofibroblasts at the later stage. Loss of β-cell mass was then found to result from TGFβ1-triggered epithelial-mesenchymal transition (EMT) by β-cells, rather than resulting directly from β-cell apoptosis. Mechanistically, TGFβ1-treated β-cells activated expression of the EMT regulator gene Snail in a SMAD3/Stat3-dependent manner. Moreover, forced expression of forkhead box protein O1 (FoxO1), an antagonist for activated Stat3, specifically in β-cells ameliorated β-cell EMT and β-cell loss and prevented the onset of diabetes in mice undergoing PDL. Together, our data suggest that chronic pancreatitis may trigger TGFβ1-mediated β-cell EMT to lead to CPRD, which could substantially be prevented by sustained expression of FoxO1 in β-cells.

Prion protein is essential for diabetic retinopathy-associated neovascularization

Diabetic retinopathy (DR), a major complication of diabetes caused by vascular damage and pathological proliferation of retinal vessels, often progresses to vision loss. Vascular endothelial growth factor (VEGF) signaling plays a pivotal role in the development of DR, but the exact underlying molecular mechanisms remain ill-defined. Cellular prion protein (PrPc) is a surface protein expressed by vascular endothelial cells, and the increased expression of PrPc is associated with physiological and pathological vascularization. Nevertheless, a role for PrPc in the development of DR has not been appreciated. Here, we addressed this question. We found that the development of streptozocin (STZ)-induced DR, but not the STZ-induced hyperglycemia/diabetes itself, was significantly attenuated in PrPc-KO mice, compared to control wildtype (WT) mice, evident by measurement of retinal vascular leakage, retinal neovascularization, a retinopathy score and visual acuity assessment. Moreover, the attenuation of DR severity seemingly resulted from attenuation of retinal neovascularization via VEGF/ras/rac signaling. Together, our study suggests a previously unappreciated role for PrPc in the development of DR.